Life as a Microscopist
- Q & A with Igor Siwanowicz
“Life as a microscopist” is a series about men and women behind the microscopes. I was very honored to exchange a few messages with Igor Siwanowicz - scientist, photographer and microscopist - who gives us some insight into the biology of the tiny organisms he likes to study, and how he became interested in them and in microscopy. It was all so interesting, I kept everything. Enjoy.
1) About your winning entry at the Nikon Small World: can you tell us more about that organism?
Aquatic bladderworts prefer clean, nutrient-poor ponds and lakes; they satisfy their nitrogen needs by trapping minute prey – water fleas, copepods, rotifers etc. – in specialized organs called bladders, which are considered the most sophisticated trapping organs in the plant kingdom, a true testimony to evolution’s ingenuity.
Finding the specimen was one of those serendipitous events – I stumbled upon bladderwort while collecting dragonfly nymphs for my research in a pond located few miles away from my institute (Janelia Research Campus of HHMI in Ashburn, Virginia). Perhaps it is the perversion of the role reversal when a plant is devouring an animal that makes flesh-eating plants so interesting; I have been fascinated with carnivorous plants since early childhood – watching “Little Shop of Horrors” might have something to do with igniting my fascination. Admittedly, bladderwort is – at the first glance - far less spectacular than, say, a Venus flytrap or a pitcher plant; when magnified though its trap reveals amazing complexity. I had a very fruitful run with this plant – with the samples I collected I was able to produce a series of images, and several of them won prizes.
This pictures depicts the awesomeness of the bladderwort trapping organ and scored the 1st place in 2013 Olympus Bioscapes contest. The image shows the inside of a trap of the aquatic carnivorous plant, humped bladderwort (Utricularia gibba). Several elements of the bladder’s construction are visible in the image, giving some insight into working of this tiny – only 1.5 mm long – but elaborate suction trap. The driving force behind the trapping mechanism is hydrostatic pressure: the plant “cocks” the trap by pumping water out of the bladder, accumulating potential energy in its thick and flexible walls like in the limbs of a bow. Specialized cells called bifid and quadrifid glands are responsible for the task of active transport of water. They line the inner walls and are visible in the image as bright-blue elongated shapes. An unsuspecting prey – usually a tiny aquatic arthropod – is guided toward the trapdoor by antenna-like branches surrounding the entrance. Quite literally, the trap has a “hair trigger” – touching one of the trigger hair cells extending from the bottom of the trapdoor (their bases are visible right in the center of the upper half as 4 small bright circles; they are much better visible in the Nikon contest image where you can see the entrance to the trap (or the bladderwort’s “mouth”)) causes the entrance – or “valve” – to bulge inward. Once the equilibrium is disturbed, the walls rapidly spring back to their initial position and the prey is sucked in within a millisecond (1/1000th of a second), experiencing acceleration of 500 G! In the image, the valve resembles a mosaic-covered Byzantine arch; it is made of a single layer of tiny, densely packed cells regularly arranged in concentric fashion. Once inside, the prey dies of anoxia and is digested by enzymes secreted by the bifid and quadrifid glands.
The intricately shaped objects visible in the lower part of the image are those aforementioned green algae called desmids; two species belonging to the genus Micrasterias and three species of Staurastrum can be identified. Various authors have described algae in Utricularia traps as commensals (algae that thrive and propagate in the nutrient-rich interior of the trap), symbionts (bladderwort benefits from the carbohydrates produced by algae) or as prey. Recent studies show that algae are able to survive only inside older, inactive traps; more than 90% are killed inside vivid, young traps. It may be that late in the season when I collected the specimens, most of the traps were already inactive, which could explain why the trapped desmids seemed to be doing fine.
2) How did you start in the field of microscopy? Do you think that microscopy can be considered a form of art?
My interest in natural sciences and nature photography were developing simultaneously - my parents are biologists and I grew up surrounded by biology textbooks. I enjoyed browsing through the illustrations and photographs long before I learned how to read. It wasn’t until 14 years ago – 2 years into my PhD studies - that I bought my first camera and found myself on the supply side of nature photography, with the special focus on macro technique. I quickly realized that microscopy would perfectly complement that activity and give me an even more intimate perspective of my “models.”
Six years ago - after abandoning protein biochemistry and moving to the field of neurobiology - I finally gained an access to a confocal microscope. For the past four years I’m spending most of my working hours imaging various bits of invertebrate anatomy – mostly dragonflies, since that is our group’s model organism. In this way I managed to merge my extracurricular expertise of macro photography and insect anatomy with scientific approach.
Although it is not the primary objective of scientific visual data, surprisingly many research-related images have aesthetic merit; to fully appreciate the beauty of those often abstract and surreal forms one needs to approach them with an open mind. A French polymath and philosopher of science Jules Henri Poincare said that “the scientist does not study nature because it is useful; he studies it because he delights in it, and he delights in it because it is beautiful”. Not many scientists these days have the privilege and comfort to apply this somewhat utopist approach to their research, but lots do share the appreciation of beauty and are fully aware of the aesthetic aspects of their work. The marriage of scientific approach and artistic talent can be best exemplified by awe-inspiring work of Ernst Haeckel, who’s “Artforms from Nature” is a continuous source of inspiration for me.
Olympus And Nikon contests are organized with such people in mind. Images are rewarded for the artistic merit and visual aspects on par with and often above their scientific importance; that definitely grants those contests a broad appeal among non-experts and contributes to redeeming the image of science as a somber, wonder-less, unexciting affair utterly unintelligible for a layperson.
A bit about sample preparation and data collecting:
Back in the laboratory I embedded isolated traps in agar-agar gel and cut them into 0.5 mm-thick slices with a vibrating razor blade. Due to the chance component inherent to the process, in only 6 out of two dozen or so specimens, the razor passed either through the midline to produce two nearly equal halves, or through the plane parallel to the bladder’s trapdoor – a satisfactory success rate.
To produce the image, I used a laser scanning confocal microscope, a device that collects images in a very different way than a brightfield microscope (your standard biology class microscope). The confocal microscope is a fluorescent microscope; it means that the imaged specimen is illuminated (excited) with light of certain wavelength and emits light of a different, longer wavelength. The source of the excitatory light is a laser; a confocal microscope can be equipped with several lasers producing light of different frequencies (i.e., wavelength, or simply color), since each fluorescent molecule (a pigment that emits light) used in research only absorbs certain specific wavelengths of light. The specimen is illuminated, point by point, by a focused laser beam that moves somewhat like an electron beam producing the familiar scanned image on the phosphorescent surface of a cathode-tube TV or computer monitor. The light emitted from the specimen is collected by the objective and passes through a pinhole aperture that cuts off stray rays of light arriving from fragments of the sample that are not in focus – only light that is emitted from the very thin area (optical slice) within the focal plane can pass. Emitted light is then detected by the microscope’s photodetector (photomultiplier), and the image is reconstructed – point by point – on the computer screen. Because most specimens are much thicker than the focal plane, a series of images - called a “stack” - is collected by moving the specimen up or down. From those images, a three-dimensional image of the sample can be reconstructed.
In most cases samples have to be made fluorescent by the use of dyes or conjugated antibodies specifically binding certain intra- or extracellular structures. To be able to image cellulose (building material of plant’s cell walls) I used Calcofluor White, adye first used in the textile industry for its propensity for binding cellulose fibers but then abandoned because of its toxicity; Calcofluor still finds use in medicine for identification of fungal pathogens in animal tissues.
A confocal microscope “sees” the sample very differently than we do - to our eyes the specimens appear very different than the final image. The amount of ultraviolet light in sunlight is – fortunately! – too low to appreciably excite Calcofluor, and all we see is green from the natural pigment chlorophyll. When illuminated with UV light (405 nm), the dye present in cell walls glows bluish-green. The same short wavelength light is absorbed by chlorophyll, which emits red light.
To produce the image, I recorded emission in three channels (colors) simultaneously. Assignment of the color in the captured image to any given channel is purely arbitrary; however, I do assign blue to the channel recording light of the shortest wavelength, green and red in similar fashion, in the “natural” order. Combining the three channels - three prime color images – into one produces the whole palette of colors – in effect, it is like the microscope had trichromatic “vision,” just as we do.
First and second photo are of barrel-shaped diatoms. Diatoms aren’t the typical green color of algae. Instead, they have brown or yellow chloroplasts. They excrete a mucus-like slime and when in large colonies are sometimes referred to as “rock-snot”. They can also cloud up the walls of fish tanks, leading people to believe they are bacteria due to the brown color of the colonies.
Third photo is Micrasterias, which is a lucky find since they seem to be quite rare where I live
Last photo is a Cladophora filament, which is the algae that makes up those adorable marimo moss balls. A few protists have made it their home.
This composite image of a single-celled freshwater algae took third place in the 2013 Olympus BioScapes digital imaging competition. Dr. Igor Siwanowicz, a neurobiologist with the Howard Hughes Medical Institute, took the image of the symmetric microbes within a family of eukaryotic green algae called desmids.
He used confocal microscopy and magnified the organisms 400 times, creating a single composite image that contains seven different species. The species pictured are Micrasterias rotata, Micrasterias sp., Micrasterias furcata, Micrasterias americana, Micrasterias truncata, Euastrum sp. and Cosmarium sp.